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The ends of linear eukaryotic chromosomes, telomeres, distin-guish natural chromosome ends from DNA double stranded breaks and thus promote genome stability. Telomeres comprise a repetitive DNA skeleton, which is wrapped in specific protein complexes. Recent data indicate that an additional building block of telomeres is RNA and that the longstanding idea that telomeres are silent genomic regions needs to be overturned. Mammalian telomeres are indeed transcribed into RNA molecules, which remain associated with telomeric chromatin, suggesting RNA-mediated mechanisms in organizing telomere architecture.

Telomeres fulfill essential functions for chromosome stability by protecting chromosome ends from inappropriate DNA repair and DNA degradation.1-4 The mammalian telomere core comprises tandem arrays of duplex 5'-TTAGGG-3' repeats (with the G-rich strand extending beyond its complement to form an overhang), bound to a multiprotein complex known as shelterin, which assures proper telomere length regulation and telomere protection.1-4 Human shelterin comprises the six factors TRF1, TRF2, TPP1, POT1, TIN2 and hRap1, while mouse shelterin is composed of seven factors due to a recent gene duplication event in the rodent lineage, which led to formation of two POT1 paralogs (POT1a and POT1b).2,5-7

In mammals, most adult somatic cells lack telomere lengthening mechanisms and their telomeres progressively shorten with each cell division cycle due to the inability of the replicative DNA polymerases to replicate the most 3' part of linear DNA molecules and due to nucleolytic processing of telomeres.1,3,8,9 Upon continuous short-ening, telomeres eventually reach a critical length (critically short telomeres) and they become recognized by the cell as double stranded DNA breaks, triggering an irreversible cell cycle arrest known as cellular senescence.10-14 The immortal potential of cancer cells depends on the reactivation of telomere lengthening mechanisms, which counteract telomere sequence loss. In this light, telomere driven cellular senescence represents one of the most powerful cellular barriers against cancer development and progression.15,16

The majority (8590%) of all human tumors maintain telomere length through reactivation of telomerase, a specialized reverse tran-scriptase, composed of a catalytic subunit and an RNA moiety, able to add telomeric repeats to the telomeric 3' end.1,3,4,17-20 In a small class of soft tissue sarcomas with complex karyotypes telomerase is not reactivated and telomeres are maintained by the so-called ALT (ALTernative lengthening of telomeres) mechanism, which involves homologous recombination between telomeres.21

Based on chromosome staining procedures, telomeres have been classified as constitutive heterochromatic domains for several decades.22 Indeed, we now know that mammalian telomeres are enriched in heterochromatin marks including histone H3 trimethyl-ated at lysine 9 (H3K9m3), histone H4 trimethylated at lysine 20 (H4K20m3) and Heterochromatin Protein 1 (HP1).23-25 Similarly, mammalian subtelomeric regions (the chromosomal regions adja-cent to telomeres) display high levels of DNA methylation, another heterochromatic signature.23,26 The heterochromatic state of telo-meres and their gene-less nature suggested the idea that telomeres are transcriptionally silent genomic regions. Supporting this notion, reporter genes experimentally inserted in subtelomeric regions undergo gene silencing, in a process known as telomere position effect.27-30

TERRA: A Regulated Nuclear RNA Transcribed from Telomeres

Contrary to the idea of telomeres being silent genomic regions, we have discovered that mammalian telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) molecules, which constitute a novel class of mammalian RNAs.31 More recently, Schoeftner and Blasco also reported on the existence of TERRA.32 Mammalian TERRA molecules contain UUAGGG repeats (while CCCUAA-contaning RNA seems not to be present in mammalian cells at detectable levels), range in size from about 100 bases up to >9 kilobases and are detected exclusively in nuclear fractions.31,32 TERRA is present in different human cell lines as well as in mouse, hamster and, outside mammals, in zebrafish cells indicating that TERRA is evolutionarily conserved in vertebrates.31,32 Using Northern blot and RT-PCR analysis we were also able to demonstrate that not only human telomeres but also subtelomeres are transcribed and that at least a fraction of TERRA molecules comprise subtelomeric-derived RNA and UUAGGG repeats.31 Thus, TERRA transcription starts from different subtelomeres towards chromosome ends. Further

analysis will be necessary to map TERRA transcrip-tion start sites and promoter regions.

While the length distribution of TERRA molecules seems not to vary substantially among the different mammalian cell lines, TERRA abundance is variable, suggesting cell type- and species-specific TERRA regu-latory mechanisms.31,32 Several lines of evidence seem to indicate that TERRA abundance might positively correlate with telomere length; for example HeLa cell lines bearing very long telomeres (HeLa-E1 and HeLaI.2.11) show higher TERRA levels as compared to HeLa cell lines with much shorter telomeres.31,32 Nevertheless, mouse RenCa cells, which have 46 kb long telomeres, display TERRA amounts similar to the ones detected in mouse embryonic fibroblasts (MEFs) with telomeres longer than 23 kb; similarly, mouse NS1 cells and mouse ES cells display similar TERRA levels while their telomere lengths are substantially different.31,32 However, it may be difficult to establish a connection between TERRA levels and telomere length when using non-isogenic cell lines because of possible cell line specific variations in TERRA regulatory mechanisms. Interestingly, Schoeftner and Blasco show that TERRA levels are lower in MEFs deficient for telomerase as compared to wild type cells, suggesting that telomere length might correlate with TERRA expression and/or telomerase might be directly involved in regulating TERRA expression. However, TERRA is present both in telomerase-positive and negative cells.31,32

TERRA is detectable in most adult mouse tissues (Fig. 1A and ref. 32) and in skeletal muscle at different developmental stages from E17 embryonic stage (Fig. 1B). As for different cell lines and species, TERRA abundance varies among the different tissues and different developmental stages. Interestingly, low TERRA levels are detected in some cancerous tissues as compared to healthy tissues, suggesting that TERRA might be downregulated during cancer development in vivo.32

Experiments performed using the RNA polymerase II (RNA polII) specific inhibitor alpha-amanitin demonstrated that TERRA molecules are at least in part produced by RNA polII-mediated transcription of mammalian telomeres. Consistently, RNA polII could be localized to telomeres by chromatin immuno-precipitation experiments and was reported to physically interact with the shelterin component TRF1.32 Schoeftner and Blasco also showed that TERRA molecules can be recovered in polyA+ enriched RNA fractions from mouse and human cell lines, suggesting that TERRA is 3' polyadenylated.32 However, we only find 7% of TERRA molecules in polyA+ nuclear RNA fractions from human HeLa cells (Fig. 2), while 6264% of c-myc and actin mRNA molecules could be detected in polyA+ fractions in our experimental conditions (Fig. 2). As expected, only a negligible fraction (1%) of non-polyadenylated U1 RNA was detected in polyA+ fractions. Notably, polyA+ TERRA molecules were almost exclusively larger

than 2 kb in length (Fig. 2) indicating that short TERRA molecules are not polyadenylated. Nonetheless, it is important to point out that the information about the sequences of the subtelomeric tract of TERRA is very limited and that one cannot formally exclude that internal polyA rich sequences also contributed to the retention of TERRA on the oligo dT column.

TERRA is a Constitutive Component of Telomeric Heterochromatin

RNA fluorescence in situ hybridization experiments revealed that TERRA molecules form discrete nuclear foci overlapping with telomeric heterochromatin in interphase cells as well as in tran-

Figure 1. (A) RNA from different adult mouse tissues immobilized on nylon filters (Zyagen Laboratories, San Diego, USA) was hybridized to radiolabelled (CCCTAA)n probes.31 Actin was used as a loading control. (B) RNA from skeletal muscles from mice at different develop-mental stages (Zyagen Laboratories, San Diego, USA) was hybridized as in (A). E, embryo; d, day; w, week; m, month. (C) HeLa cells were treated with 100 nM Trichostatin A (TCA) for 24 h or left untreated (untr). Total RNA was prepared and subjected to Northern blot analysis as in (A). Actin was used as a loading control.

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Transcription of telomeric heterochromatin

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scriptionally inactive metaphase chromosomes.31,32 The ability of TERRA molecules to interact with telomeres independently on their transcription indicates that TERRA is an integral component of telomeric heterochromatin and suggests that TERRA might partici-pate in maintaining telomere architecture. It is possible that TERRA regulates the epigenetic state of telomeres, perhaps by mechanisms similar to the inactivation of the mammalian X chromosome in females, which is mediated by the long noncoding RNA Xist.33,34 Intriguingly, some TERRA foci at least partially overlap with inactive X chromosome domains.32

TERRA Regulatory Pathways

The recent discovery of TERRA was also accompanied by the discovery of different pathways involved in the regulation of either TERRA abundance or cellular localization. A careful dissection of such pathways will help us to decipher TERRA associated functions as well as to clarify the general role of non-coding RNAs in modula-tion of transcription and in heterochromatin formation.

endonuclease hEST1A (or SMG6) and the PI3-kinase-like kinase SMG1 promote genome stability by mechanisms separated from their function in NMD.36-39 We have shown that a fraction of all seven human SMG proteins associate with telomeres in vivo and that short hairpin RNA (shRNA) mediated depletion of different SMG proteins induces accumulation of telomere associated TERRA molecules, without affecting TERRA degradation rate or total cellular levels.31 Thus, SMG proteins promote TERRA displace-ment from telomeres. Interestingly, while depletion of UPF1, hEST1A or SMG1 drastically impaired with TERRA displacement from telomeres, only a mild effect was obtained when cells were depleted for the other SMG factor UPF2.31 Because similar impair-ment of NMD was observed when UPF1, hEST1A, SMG1 or UPF2 were depleted, it seems conceivable that different SMG proteins play differently important roles in modulating TERRA association with chromatin.31 In this respect, the reported enzymatic activi-ties of UPF1 and hEST1A may be largely responsible for TERRA regulation. Human UPF1 is a 5...